After injury, unlike the axons of the central nervous system where the regeneration process is poor or absent, axons of the peripheral nervous system are able to regenerate, though inefficiently. Uncovering the molecular mechanisms behind axon regeneration will be of great value for the medical handling of traumatic nerve injuries and will also offer valuable insights regarding many neurodegenerative diseases. The use of large vertebrate animal models for gene identification requires complex assays and instrumentations. On the other hand, thanks to their simplicity and large similarities to mammals, the use of small invertebrate animals is of great interest for rapid genome-wide screenings. Among these animals, the nematode Caenorhabditis elegans (C. elegans) is one of the most powerful model organisms providing a wide-range of genetic tools. It is especially versatile to forward and reverse genetics, whether phenotypes of a given gene are investigated or genes involved in a specific phenotype are. However, until recently, the use of C. elegans for nerve regeneration studies was limited due to a lack of precise surgical techniques for axotomy. Two of our recent technological innovations, ultrafast laser nanosurgery and microfluidics immobilization have made it feasible to study axonal regeneration in this genetically tractable model organism. While the ultrafast laser nanosurgery has finally enabled us to severe axons of this small worm with high precision, the microfluidic immobilization chip has enabled rapid trapping of worms for surgery without using any anesthetics, reducing the time required to perform the surgery by a factor of 100 (from tens of minutes to several seconds) and eliminating the possible side effects of the anesthetics. Such microfluidic chip finally offers the possibility to perform high-throughput screening, provided that large sample populations can be automatically loaded instead of manual handling. The goal of this research project is threefold: (1) fabricate a microfluidic device that can automatically deliver worms from multi-well plates to the microfluidic axotomy chip, (2) develop its computer assisted automation, and (3) demonstrate the performance of the integrated system for rapid screening of candidate genes affecting axonal regeneration in C. elegans by RNA interference (RNAi). This novel device will facilitate the manipulation of large population samples for delivery to the axotomy chip and for their storage after the axotomy for further study of the regeneration results. Development of such a high-throughput screening platform requires integration of different modules for RNAi feeding, nanosurgery, recovery, and imaging and their synchronization through computer controlled automation. A platform capable of handling 1000's of worms from individually addressable wells will facilitate any automated screening studies, thus greatly reducing time and cost. PUBLIC HEALTH RELEVANCE: To accelerate large-scale screening of C. elegans, we propose to engineer a novel microfluidic multiplexer to interface with standard well-plates (microtiters) for transferring worms automatically and precisely from individual wells into different imaging and surgery microfluidic modules. The successful automation of this microfluidic multiplexer will allow us to perform high throughput screening of genes affecting nerve regeneration using RNAi interference and femtosecond laser nano-axotomy. Owing to the genetic similarity between human and C. elegans, a better understanding of the molecular mechanisms underlying nerve regeneration in the worms will eventually enable the development of treatments and preventions of human degenerative diseases.